杀线虫芽孢杆菌B16和枯草芽孢杆菌168硫酯酶Ynep差异分析

Comparison of thioesterase Ynep in Bacillus nematocida B16 and Bacillus subtilis 168

摘要:

【背景】线虫生防菌杀线虫芽孢杆菌B16和可作为线虫食物的枯草芽孢杆菌168对线虫存在差异感知，线虫引诱剂2-庚酮的表达量也存在显著差异。【目的】研究线虫生防菌芽孢杆菌B16和可作为线虫食物的枯草芽孢杆菌168中合成甲基酮的关键因子硫酯酶Ynep，并分析它们的异源表达、结构及酶活性质上的差异。【方法】使用定量聚合酶链式反应（quantitative PCR，qPCR）验证两种芽孢杆菌中硫酯酶在转录水平的差异，通过生物信息学分析两种酶结构的差异，并通过蛋白异源表达和活性测试验证其蛋白表达水平及特性的差异。【结果】qPCR显示，在培养12 h和72 h后，菌株B16 ynep基因表达分别为菌株168的1.428和0.991倍。生物信息学分析表明两者氨基酸序列一致性为97.12%，酶分子量分别为15 993.35 Da和16 007.42 Da，等电点（isoelectric point，pI）分别为7.06和7.82，均具有与转录过程中基因表达调控有关的蛋白质（transcriptional enhancer，TE）结构域，均包含保守的Asp16-His23-Tyr26催化三联体。同源建模显示Ynep均呈典型“hot dog”结构，是TE超家族最常见的结构折叠。两种酶存在4个氨基酸差异，可能影响硫酯酶四聚体形成。菌株B16和168 Ynep与底物β-酮酯酰-CoA分子对接，结合能分别为—5.47 kcal/mol和—6.95 kcal/mol。异源表达纯化的酶活性测试显示：菌株B16和168的酶活分别为1.243 U/mL和1.233 9 U/mL，最适反应温度均为30 ，最适pH分别为8.0和9.0。两种酶在10—30、pH 7.0—8.0均表现出良好的稳定性，且菌株168中Ynep对温度耐受性更佳。【结论】两种芽孢杆菌中的硫酯酶Ynep在异源表达、结构及酶活性质方面均有差异，导致其合成线虫引诱剂2-庚酮量有所不同。这些研究结果有助于解析不同芽孢杆菌差异感知线虫的分子机理，为进一步开发高效新型线虫生防产品提供新思路。

Abstract:

[Background]The biocontrol bacteria Bacillus nematicida B16 and B. subtilis 168 present different perceptions for nematodes and have significant differences in the expression of the nematode attractant 2-heptanone.[Objective] To investigate the key factor, the thioesterase Ynep, involved in methyl ketone synthesis in B. nematicida B16 and B. subtilis 168, and compare the heterologous expression, structure, and activity of this enzyme between the two strains. [Methods] Quantitative polymerase chain reaction (qPCR) was employed to examine the transcriptional levels of thioesterase in the two Bacillus strains. Bioinformatics analysis was conducted to compare the structural differences of this enzyme. Furthermore, the heterologous expression level and activity of this protein were compared between the two strains. [Results] The transcription level of ynep in B. nematicida B16 was 1.428 and 0.991 folds of that in B. subtilis 168 at the time points of 12and 72 h, respectively. Bioinformatics analysis indicated an amino acid sequence identity of 97.12% between the two enzymes. The two enzymes were predicted to have the molecular weights of 15 993.35 Da and 16 007.42 Da and the isoelectric point (pI) values of 7.06 and 7.82, respectively. They possessed the transcriptional enhancer (TE) domain with the conserved catalytic triad Asp16-His23-Tyr26. Homology modeling revealed that both Ynep enzymes presented the typical "hot dog" fold, a common structure in the TE superfamily. Four residue differences existed between the two enzymes, potentially influencing the formation of the thioesterase tetramer. The Ynep in B. nematicida B16 and B. subtilis 168 showed the binding energy of -5.47 kcal/mol and -6.95 kcal/mol, respectively, with the substrate β-ketoacyl-CoA. The purified recombinant enzymes of B. nematicida B16 and B. subtilis 168 demonstrated the activities of 1.243 U/mL and 1.233 9 U/mL, respectively. Both enzymes exhibited optimal reaction temperatures of 30 and optimal pH 8.0 and 9.0, respectively. Both enzymes demonstrated good stability within the temperature range from 10 to 30 and at pH 7.0-8.0. Moreover, the Ynep in B. subtilis 168 had stronger thermal tolerance than that in B. nematicida B16. [Conclusion] The thioesterase Ynep in the two Bacillus strains shows differences in heterologous expression, structure, and enzyme activity, which lead to variations in the synthesis of 2-heptanone. The findings contribute to the understanding of the molecular mechanisms underlying the different perceptions of nematodes by different Bacillus strains, providing new insights for the development of efficient biocontrol products against nematodes.